Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency.
The first practical test for the determination of creatinine in urine, known as the Jaffe method, involves the formation of the red-yellowish brown colored creatinine picrate by the bonding of picric acid and creatinine in an alkaline solution. A more recent method for creatinine determination is reported by Benedict and Behre J. Biol. Chem., 113:515 (1936) which involves the reaction of 3,5-dinitrobenzoic acid with creatinine in an alkaline medium.
Each of these reactions require a high pH, i.e. on the order of 12-13, in order to deprotonate the creatinine so that the system can operate properly. Strongly basic substances such as alkali and alkaline earth metal hydroxides are typically used to maintain a suitably high pH in these reagent systems. This, however, presents several problems when an absorbent carrier such as filter paper or a porous film is used as carrier for the reagent system because upon introduction of the alkali, the carrier tends to become brittle and it is difficult to obtain even distribution of the alkali throughout the carrier matrix. Furthermore, when the reagents are applied to the carrier in the form of a solution whereupon the solvent is evaporated to leave a dry residue, the dried alkali does not readily solubilize when contacted with a fluid such as urine which is being examined for creatinine concentration.
In Japanese Patent Application No. 62-287261 there is disclosed a method for facilitating the application of alkaline ingredients to absorbent carriers which involves dipping the carrier into an aqueous solution of the alkali and an alkali soluble carboxylic acid derivative polymer followed by application from an organic solvent of a dye precursor which develops color upon reacting with creatinine under alkaline conditions.